The main objectives of the proposal, detailed in specific aims la-d and 2a-c are to understand the molecular logic of the multimedia assembly lines for the biosynthesis of no ribosomal peptide antibiotics. Nonribosomal peptide synthetase (NRPS) assembly lines to be analyzed include the antibiotics novobiocin and clorobiocin, tyrocidine and gramicidin, the immunosuppressant rapamycin the phytotoxins coronatine and syringomycin from Pseudomonas syringae, and the antitumor drug candidate epothilones. All these medicinally active natural products are built up as acyl chains via initiation, elongation, and termination NRPS modules. This proposal focuses on initiation (specific aim 1a-d) and termination (specific aim 2a-c) module strategies. Specific aim 1 addresses the logic, organization, and catalytic specificity of free standing A-T didomain subunits acting as initiation modules in nontraditional NRP assembly. This includes the early steps that form the bicyclic aminocoumarin scaffold of novobiocin as well as the late stage reactions that generate the 5-methylpyrrolycarboxyl moiety that interacts with the ATP site of the GyrB subunit of DNA gyrase. It also focuses on companion 2 His/Asp- Fe (11)enzymes proposed to be cyclopropanation catalyst (coronamic acid formation) or chlorination catalyst (4-chlorothreonine in syringomycin). Specific aim 2 explores the molecular logic for catalytic macrocyclization by the C-terminal domains of NRPS and hybrid polyketide/NRP assembly lines. Aim 2a deals with the range of macrolactamization by the Thioesterase (TE) domain excised from the tyrocidine syntherase assembly line, while aim 2b analyzes the cognate TE at the end of the EpoF subunit of the polyketide synthase for the 16 membered epothilone macrolide. Specific aim 2c examines the hybrid PK/NRP assembly line for rapamycin where the most C terminal domain in the RapP subunit is a Condensation (C) domain, not a TE domain, yet is thought to form the 34-membered macrolactone ring in both rapamycin and the cognate FK506 immunosuppressants. Deciphering the molecular logic of both chain initiation and chain termination strategies may facilitate subsequent efforts directed at reprogramming the initiation and termination machinery to make novel variants with altered and improved therapeutic activities.